Vascular calcification is a frequent complication of vascular disease associated with increased morbidity and mortality. It is recognized to be an active process involving osteochondrogenic differentiation in multipotent cells. Previous reports have suggested that endothelial cells (ECs) contribute calcifying cells in congenital disorders with soft tissue calcification, triggered by abnormal bone morphogenetic protein (BMP) signaling. Matrix Gla protein (MGP) is an inhibitor of vascular calcification, and an antagonist to BMP2/4. MGP null mice show strong activation of vascular BMP signaling and extensive aortic calcification. Ins2-Akita/+ mice, a model of type 1 diabetes, exhibit similar findings due to induction of BMPs and BMP receptors. Preliminary data show that BMP activation causes stem cell characteristics to emerge in normal ECs. Indeed, EC lineage markers co- stain with osteogenic and pluripotent stem cell markers in MGP null aortas, and MGP-depletion in human aortic ECs in vitro causes the cells to undergo osteogenic differentiation in response to osteoinductive factors. In addition, Ins2-Akita/+ mice exhibit increased aortic expression of multiple pluripotency-associated markers. Thus, normal ECs may contribute osteoprogenitor cells to vascular calcification when subjected to increased BMP activity. The scientific purpose of this proposal is to define the role of the endothelium in vascular calcification in MGP deficiency and diabetes. We hypothesize that normal endothelium contributes multipotent cells to vascular calcification due to the capacity of abnormal BMP activity to induce stem cell characteristics and subsequent osteogenesis. Specific Aim 1 will define multipotency and osteochondrogenic potential in ECs in the setting of MGP deficiency or hyperglycemia in vitro and in vivo. Specific Aim 2 will determine if ECs contribute cells to calcific lesions using lineag tracing in two models of vascular calcification, the MGP-/- mouse and the diabetic Ins2Akita/+ mouse. For lineage tracing, the currently available Tie2-GFP or Tie2- Cre;R262R-eGFP transgenic mice will be used. Specific Aim 3 will determine the relative contributions of EC- and SMC-specific loss of MGP in the development of vascular calcification using EC- and SMC-specific MGP knockout mice. If successful, the endothelium may emerge an important target to treat calcification in acquired vascular disease.